Lignocellulose is the most abundant biopolymer on earth. Lignocellulose is the major structural component of woody plants and non-woody plants such as grass. Lignocellulosic biomass refers to plant biomass that is composed of cellulose, hemicellulose, and lignin. Large amounts of lignocellulosic residues are produced through forestry, timber and pulp and paper industries and agricultural practices (e.g. straw, stover, sugar cane bagasse, chaff, hulls) and many agroindustries. Also municipal waste contain fractions that can be considered as lignocellulose residues, such as paper or cardboard waste, garden waste or waste wood from construction. Lignocellulosic residues, such as agricultural residues, offer highly sustainable, non-food and non-ILUC (indirect land use change), alternative for production of biofuels. In addition, due to high abundance and low price lignocellulosic residues are preferred materials for production of biofuels. In addition, dedicated woody or herbaceous energy crops with biomass productivity have gained interest as biofuel use.
The production of biofuels, especially ethanol, from lignocellulosic materials by microbial fermentations has been studied extensively. The greatest challenge for utilization of lignocellulosics for microbiological production of biofuels or biofuel feedstocks lays in the complexity of the lignocellulose material and in its resistance to biodegradation. In lignocellulose, cellulose (20-50% of plant dry weight) fibers are embedded in covalently found matrix of hemicellulose (20-40%), pectin (2-20%) and lignin (10-25%) forming very resistant structure for biodegradation. Further, the sugar residues of hemicellulose contain a varying mixture of hexoses (e.g., glucose, mannose and galactose), and pentoses (e.g., arabinose and xylose) depending on the biomass.
Certain microorganisms can produce lipids from organic molecules, such as sugars derived from lignocellulose. Certain microorganisms, typically yeast, fungi or bacteria, can efficiently convert both C6 and C5 sugars in lignocellulosic materials to oil. Oil produced by heterotrophic microorganisms is often called as single cell oil or microbial oil. Single cell oil production process using heterotrophic microorganisms typically comprises cultivating microorganisms in aerated bioreactors, allowing cells to accumulate lipids, harvesting lipid-rich cells and recovering oil from cells. Microorganism-based lipids (i.e. single cell oils) can be used as raw materials for production of biofuels such as biodiesel, renewable diesel or bio jet fuel.
The economically feasible production of biofuels from lignocellulosic materials by microbial fermentation requires an efficient conversion of all the main carbohydrate constituents of the lignocellulosic materials to biofuels. On the other hand the economic feasibility of the biofuel production requires that all the main carbohydrate constituents of the lignocellulosic material have to be converted to sugars, which are suitable for microbial production. Generally this means breaking (hydrolyzing) the polymeric structures of hemicellulose and cellulose to obtain monomeric sugars.
The prior art discloses several methods, which can be used for production of sugars from lignocellulosic materials.
Patent publication US2008/032344 A1 discloses a process for recovery of cellulosic sugars and near native lignin co-product from lignocellulosic biomass. The process comprises subjecting the raw material to autohydrolysis, organosolv and enzymatic hydrolysis treatments to produce a cellulosic sugar solution comprising glucose, which is fermented with yeast and/or appropriate recombinant organism to produce biofuel and/or chemical.
Cunningham and Carr (1984) have reported various technologies to remove hemicellulose and lignin from wheat straw to provide an upgraded cellulosic residue for enzymatic hydrolysis. They have also disclosed a pre-treatment method comprising autohydrolysis of the wheat straw followed by subsequent alkali treatment with NaOH. The amount of NaOH used in delignification of autohydrolyzed wheat straw is significantly higher than in the present invention. According to the teachings of Cunningham and Carr the delignification treatment of autohydrolysed straw did not significantly improve the cellulose conversion to glucose in the enzymatic treatment step whereas alkali treatment without autohydrolysis step significantly improved the results of the enzymatic hydrolysis (see Tables III and IV). Furthermore the obtained hemicellulose and cellulose hydrolysates are not used in production of single-cell oil as in the present invention.
Patent publication US 2013/143285 A1 describes a process for conversion of lignocellulosic feedstock to fermentable sugars. The process comprises subjecting the lignocellulosic feedstock to alkali-treatment at a pH of 8-12, to a dilute acid treatment and finally to enzymatic hydrolysis to produce sugar solution comprising glucose, which is fermented to a fermentation product.
Patent publication US 2012/036768A1 describes a method for producing fermentable sugars from lignocellulosic materials, in which method a pre-treated lignocellulosic material is subjected to two-stage enzymatic hydrolysis treatment. The first enzymatic treatment comprises a mixing the pre-treated material with a first enzymatic composition to produce a first hydrolysis mixture, which is thickened to increase the fiber concentration to provide a second hydrolysis mixture. The second enzymatic treatment comprises mixing the second hydrolysis mixture with a second enzymatic composition to produce a liquid mixture containing fermentable sugars and a solid lignin phase.
One of the major challenges in production of lignocellulosic sugars from lignocellulosic material is to provide a process, which enables cost-efficient production of high quality sugar hydrolysates, which can be used without further purification in production of single-cell oil. The high quality of the sugar hydrolyzates means that the amount of impurities such as phenols and acids should be below the concentration, which is toxic to the microorganism used in the fermentation. The cost efficiency requires that the consumption of hydrolysation agents such as enzymes should be kept at low level. This can be achieved for example by recycling of the cooking chemicals. The economic feasibility requires that the quality of the side streams, which are not used as raw material for microbial fermentation, should be as high as possible to enable the valorization of these streams.
State-of-the-art lignocellulose pre-treatment technologies have been designed for anaerobic fermentations (cellulosic ethanol). Microbial oil production differs from anaerobic fermentations since it is aerobic process (requires oxygen). This invention describes a lignocellulose fractionation process that has benefits especially for aerobic bioprocesses, such as microbial oil production.